Esters of {60 -amino penicillins

ABSTRACT

New esters of Alpha -aminopenicillins having the formula   WHEREIN R is C2-C7 alkyl;   WHEREIN R2 H or OH, R3 H or halogen;   WHEREIN R1 is   OR -CH2NHCOO-R4 wherein R4 C1-C4 alkyl; R5 H, CH3 or C2H5, useful as active ingredients of pharmaceutical preparation; processes for their preparation; chemical intermediates useful in said preparation; and methods for the treatment of infectious diseases.

United States Patent Ekstr'om et al.

[111 3,873,521 1 1 Mar; 25, 1975 1 1 ESTERS OF a-AMINO PENICILLINS [73]Assignee: Astra Lakemedal Aktiebolag,

Sodertalje, Sweden 221 Filed: Sept. 7, 1971 21 App1.No.: 178,426

[30] Foreign Application Priority Data Sept. 17, 1970 Sweden .t.12688/70 Nov. 20, 1970 Sweden 15720/70 [52] U.S. C1. 260/239.l, 424/271[51] Int. Cl C07d 99/16 [58] Field of Search 260/239.1

[56] References Cited UNITED STATES PATENTS 3,674,776 7/1972 Long et a1260/2391 3,697,507 10/1972 Frederiksen et a1. 260/239.1

Primary E.\aminer-Nicholas S. Rizzo Attorney, Agent, or Firm-Brumbaugh,Graves, Donohue & Raymond {57] ABSTRACT New esters of a-aminopenicillinshaving the formula wherein R is C -C a1ky1;

wherein R H or OH, R H or halogen;

wherein R is CH3 O R O jiiiif ifiii' TFBETPTP T 14 Claims, N0 DrawingsESTERS OF oz-AMINO PENICILLINS The present invention relates to aprocess for the preparation of new esters of (Jr-aminopenicillins havingand pharmaceutical acceptable salts thereof in which formula R isselected from the group consisting of alkyl groups containing from twoto seven carbon atoms; the radical wherein R'- is selected from thegroup consisting of hydrogen and hydroxy and R is selected from thegroup consisting of hydrogen and halogen such as chlorine and fluorine;and the radicals and 3 (VII) wherein R and R have the above indicatedmeaning,

s\ o1-Il H2NCHCH wherein R has the above indicated meaning.

Salts of the new esters may be formed with inorganic acids, for instancehydrochloric acid, hydrobromic acid, hydroiodic acid or sulphuric acid,or with organic acids, for instance citric acid, tartaric acidor maleicacid. In view of the asymmetrical carbon atom in the side chain in thecompounds having the formula I, said compounds exist in two epimericforms, and the invention includes the preparation of both epimeric formsas well as mixtures thereof.

It is known that (Jr-aminopenicillins are antibiotic substances havingbroad activity spectra. Among compounds particularlya-aminobenzylpenicillin has gained a great importance, however, not onlythe antibacterial activity of a penicillin is of importance for itsclinical effect, but also its pharmacological characteristics, such asoral absorption, tissue distribution, metabolism and rate ofelimination. As regards a-aminobenzylpenicillin it has been found thatsaid compound is orally absorbed relatively incompletely.

The compounds according to the invention are well absorbed orally andresult in higher blood concentrations of the correspondingaminopenicillins than do the corresponding amounts of the latter assuch. The esters are hydrolyzed somewhat in aqueous surroundings butconsiderably more when subjected to the influence of hydrolytic enzymes,for instance such present in blood serum and other human body fluids.This property of the esters is very important, since it results in arapid release of the antibacterially .active aminopenicillins from theesters when these are absorbed from the intestinal tract or isintroduced in another manner into the flow of blood or into the tissuefluids.

Said compounds having the formula I are excellently tolerated and arepreferably taken orally, either as such or in the form of their salts,and they can be intermixed with solid carriersor adjuvants or both. Insuch preparations the ratio between the therapeutic substance and thecarriers and adjuvants may vary bet-ween l= and The preparations mayeither be processed to for instance tablets, pills or dragees or can besupplied to medical containers, such as capsules or as regards mixturesthey can be filled on bottles. Pharmaceu tically acceptable, organic orinorganic, solid or liquid carriers may be used, suitably for oral orenteral administration or for topical application, in manufacturing thepreparations. Gelatine, lactose, starch, magnesium steara'te, talc,vegetabilic andanimalic fats and oils, vegetabilic rubber andpolyalkylene gl'ykol and other known carriers for pharmaceuticals areall suitable for manufacturing preparations of said compounds. Thepreferred salt of the esters is the hydrochloride, but salts with otherinorganic or organic acids, also antibiotically active acids, may beused, for instance phosphates, acetates or salts withphenoxymethylpenicillin. Moreover the preparation may contain otherpharmaceutical active components, being suitably administratabletogether with said esters when treating infectious disceases. Forinstance other suitable antibiotical' substances.

In the treatment of bacterial infections in man, the compounds ofinvention are for example administered in amounts corresponding to 5 to200 mg/kg/day, preferably in the range of 10 to mg/kg/day in divideddosages, e.g. two, three or four times a day. They are -Continued Exam-Meth- R R Name ple d A ifibibkiadi i 6-(a-amino-fi tlnerglacetarmdo)penicillaiiate .d 31 n iL J CHOCOCH3 Same as above CH31'-eth0xycarbonyloxyethyl 6-(a-amino-3-thienylacetamido)peificillanate.B

Jar-000002115 l $Ha l -ethoxycarbonyloxy-ethyl6-(a-amino-2-thienylacetamido)penieillanato i CH-OCOOC2H5 I k r (3H31-acetoxy-ethyl 6-(a-amino-3-furylaeetamido)penicillanate 20 B MCHOCOCH:1

U CII2O C O O CzHs EthoxycarbonyloxymethylG-(a-amino-2-furylaeetamido)penicillanate.o

3-Cl-1-OI'ICaHa CH3 1-ethoxycarbonyloxy-ethyl6-(a-amino-3-chloro-4-hydroxyphenylaceta l mi d0)penici1lanate,

CHOCOOC2H v G l -CH9NHC O 0 0 11 Ethoirycarbonylaminomethylfi-(D-a-amino-m-l\uorophenylaeetamido)- penicillanate. p-FC HiCHQNHCOOCH3 Methox ifarbonylaminomethylfi-(D-a-amino-p-Iluorophenylacetamido)- pem'ci anate. O'CICQI'IJCH2NHCOOC2H5 Ethoxycarbonylaminomethyl6-(D-a-amino-o-ehlorophenylacetamido)- penieillanate.Butoxycarbonylaminomethyl 6-(D-a-aminophenylacetamido)penicilme e. C 11CHZNH C 0 0 041-19 Absorption formulations 32 Hydrolysis formulations"33 Pharmaceutical formulations 3 1 35 The preparation of compounds offormula I accordb. reacting a compound having the formula ing to theinvention is made either by a. reacting a a-substituted penicillin orsalt thereof of the formula II l A s CH: 40 n-ora-comr-ort-orr o\ (H)with an ester of 6-am1no penicillamc acid or a deriva- A CHa tivethereof according to the reaction sequence 0 ONCH-C O O R (IV) wherein Ais an ammo group, a substituted ammo /S\ /CH: group or a group that canbe transformed or converted RCHCOY 1 O (I) H to an ammo group, and R ishydrogen or a cation, l 1 with a compound having the formula IIICON-QHCOOR in which formulae R, A and R have the meaning indicatedabove, and the radicals CO- .Y and X-NH- are such radicals, that canreact with each other under formation of a CONHlinkage for the formationof compounds having the formula I, if A is an amino group, or compoundshaving the formula IV if A is thereafter converted to an amino group.

In the embodiments (a) the reaction takes place according to thefollowing reaction sequence S ()lIu ,1 R-(|)lI()ONH()H-(Jll (r v A \"lliH mm 5 It(|JII-U0Nll--H-06 (1 x- -R wherein R, A and R have the meaningindicated A above, the A-substituent of that intermediate being O:("N CHC00Y then converted to an amino group, or (II) (III) In the formulas ofthe sequence R and R have the meaning as previously indicated. A is anamino group or a substituted amino group ZNl-l or a group that can betransformed or converted to an amino group, for instance an azido ornitro group or a halogen atom, such as chlorine and bromine, COOY and XRrepresent groups that are able to react with each other under theformation of the grouping COO-R. As an example, Y may be hydrogen or analkali metal atom or a tertiary ammonium group and X may be a halogenatom, preferably chlorine or bromine, or a group functionally equivalenttherewith, alkylsulphonyloxy or aryls ulphonyloxy radical. The compoundsformed by the reaction are esters of a-A- substituted penicillins (IV).If A is the group Nl-I the formula IV represents the compounds of theinvention, whereas, when A has other meanings, the formula indicatesinteresting intermediates in the synthesis of the compounds of theinvention.

A comprehension of characteristica of substituent A is that it isselected among groups, that after the above mentioned reaction can beconverted to an amino group with methods that are sufficiently mild toavoid destruction of the molecule at the ester group or at the lactamering. Especially all such groups that have been used when synthetizingaminopenicillins are useful. Thus, the substituent A preferably has theformula ZNH, wherein Z is a benzyloxycarbon'yl group, a parahalogen,paranitro or paramethoxybensyloxycarbonyl group, a B, B,B-trichloroethyloxycarbonyl group or Z may be a sulphur-containingradical, for instance a trithylsulphenyl group, or an arylsulphonylgroup, for instance an orthonitrophenylsulphenyl group. Z may also betripehnylmethyl (also called trityl) or a group obtained by reacting thefree amino group with a B-dicarbonyl compound. such as acetylacetone, anacetic ester or bcnzoyl acetone for formation of enamines or Schiffsbases. Generally it may be said, any Z- group that can be removed byreduction, weak acid hydrolysis or by other mild reactions known per se,will be suitable The starting materials for formula II, wherein A isdifferent from NH are known as intermediates in the synthesises ofa-aminobenzylpenicillins. They exist in two epimeric forms. If thestarting materials are prepared in the form of the D- or L-epimer, thecorresponding epimeric form of the compounds of the invention isobtained. If, on the other hand, a mixture of the epimeric forms ofstarting materials is used, a mixture is obtained. If desired thismixture can be separated in the two isomers, for instance by fractionalchrystallisation. The methods for the preparation of starting materialshaving the formula II are standard methods used within the peptidechemistry and include for instance conversion of phenyl acetic acid toA- substituted phenyl acetic acid, where A has the meaning indicatedabove, followed by a reaction between a reactive derivative of saidintermediate and 6- aminopenicillanic acid, wherein the amino group maybe free or substituted, for instance with a trimethylsilyl radical. Someof the starting materials having formula II may also be prepared froma-aminobenzylpenicillin:

or salts thereof. l

The reaction of compounds having the formula II with compounds havingformula III may be carried out at or below room temperature or bycareful heating to the boiling point of the solvent used, depending onthe meaning of Y and X. It is possible to use different organic solventsor mixtures thereof with water, for instance acetone, dioxane,tetrahydrofurane, methylchloride or dimethylformamide. The reactionproducts are chrystalline or oily products, they may be used in the nextstep withoutfurther purification. By repeated precipitation the oilyproducts may beconverted to chrystalline or amorphous powders.

The subsequent reaction step (IV, I), wherein the A- group istransformed or converted to an amino group, can depending on the meaningof A be carried out by different methods khown from peptide synthesisand aminopenicillin synthesis.

Catalytic hydrogenation is preferred ,ifA has the formula ZNH-, and Zmeans benzyloxycarbonyl or closely related derivatives thereof, or if Zis trityl. The hydrogenation is preferably carried out at roomtemperature and either at atmospheric or slightly elavated pressure in asolvent that may be a non-reducing organic solvent or a mixture of suchsolvent with water. The preferred catalysts are noble metal catalysts,for instance palladium or platinum catalysts o r Raneynickel, but alsoother catalysts can be used. Electrolytic reduction can also be used inthese cases. If Z is a B, B, B-trichloroethyloxycarbonyl group,reduction with Zn in acetic acid is preferred. A weak acid hydrolyse ispreferred, if Z is a sulphur-containing radical, an enamine or a Schiffsbase, for instance a hydrolyse at a pl-I-value of about 2 in a dilutesolution of hydrogen chloride in aqueous acetone. It is also known inthe literature to remove an o-nitrophenylsulphenyl-radical by anucleophilic attack on the sulphur atom of the sulphenamide group, andin this case best yield is obtained with potassium or sodium iodide,sodium thiosulphate, sodium hydrosulfide, sodium hydrosulphite orpotassium thiocyanate. Alternatively, the sulphcnamide compound may bereacted with a thiophenol in an organic solvent, e.g. dimethylformamide.Other sulphenamide radicals are removed in the same way. If A is anazido or nitro group such groups may be converted to the free aminogroup in a manner known per se when the azido and nitro group iscatalytically hydrogenated with a noble metal catalyst or with Raney-Nickel or by electrolytic reduction. If A is a halogen atom, forinstance bromine, it is converted to an amino group by amination, forinstance with hexametylentetramine.

In the embodiment (b) a compound having formula V is reacted in a mannerknown per se with an esterof 6- amino penicillanic acid having formulaVI In these formulae R and A have the same meaning as above, andradicals COY and X l-lN are of such a nature as to be able to react witheach other under formation of a group CONH. For instance, -COY can bethe radical of an acid halide, for in- 5 stance an acid cloride or acidbromide, an anhydride, a mixed anhydride with an alkylcarbonic acid, forinstance isobutyl carbonic acid, a carbonic acid, an inor-' ganic acidor a sulphonic acid, or may be a radical obtained by reacting thea-substituted phenylacetic acid and a carbodiimide orN,N-carbonyldiimidazol or an other compound reacting in a similar way. Xmay be hydrogen or a trialkylsilyl group, wherein the alkyl group hasnot more than five carbon atoms. The reaction can be carried out in anorganic solvent or a mixture of such solvent with water, either at lowtemperature or at slightly elevated temperature. Suitable solvents aremethylene chloride, chloroform, ethylacetate, acetone, dimethylformamide or diethylacetamide, ether, tetrahydrofurane, dioxane orsimilari inert solvents. The reaction products are isolated in a.conventional manner, for instance by repeated precipi- .ation or byremoval of the solvent, followed by recrys- .allisation from a solvent.Compounds having formula V are known compounds that can be produced bystandard methods known in peptide chemistry.

The compounds having the formula VI are new compounds that areintermediates in the synthesis of compounds having formula I. They maybe prepared by reacting o-aminopenicillanic acid in the form of a salt,for instance an alkali metal salt or a triethyl ammonium salt with ahalogen alkylester having formula XR (Ill), wherein X is a halogen atom,preferably a chlorine or bromine atom, or a functionally equivalentgroup such as sulphonyloxyradical. The 6- aminopenicillanic acid can beused as such, or the 6- amino group may be protected during theesterifying process. Only protecting groups that may be easily removedwithout causing weakening of the lactame ring or the ester group aresuitable in this ease, for instance triphenylmethyl or trimethylsilylradicals. The reaction is carried out in an inert organic solvent, forinstance acetone, dimethylformamide or methylene chloride, and at orbelow room temperature or somewhat elevated temperature. If the aminogroup is protected the removal of the protecting group can becarried outin different ways, for instance by hydrogenolyse or hydrolyse underneutral or acid conditions, by which the lactame ring and the estergroup are not attacked. Reaction products having the formula VI (X H)are suitably isolated in the form of their acid addition salts with forinstance p-toluene sulphonic acid or other inorganic or organic acids,such as sulphuric acid, phosphoric acid, hydrochloric acid, acetic acid,maleic acid, tartaric acid and other similar acids.

The compounds of formula VI may also be prepared by esterifying theindustrially available penicillins or preferably their salts with acompound having the above formula Ill under similar conditions asdescribed, whereafter the side chain of the penicillin ester obtained isseparated for the formation of the a-aminopenicillanic ester having theformula VI or a salt thereof. Preferably benzylpenicillin orphenoxymethylpenicillin is used. The cleavage of the amide binding canbe carried out by means of a modification of the process described inthe Belgian Pat. No. 698,596, wherein 6-acylamino penicillanic acidester is reacted with an acid halide in the presence of an acid-bindingagent, such as quinoline or pyridine. The preferred acid halide is,however, phosphorous pentachloride, due to which the reaction in thiscase may be carried out at a low temperature, which will increase thestability of the intermediate formed, probably being an imino halide.The reaction can be carried out in different solvents,

' but the preferred solvents are chloroform and methylene chloride.

The intermediate is not isolated, but treated with a primary alcohol inexcess for formation of an iminoether. The reaction temperature andreaction time depend'on the alcohol used. In most cases temperatures beween 2 0?Caa i+ C are su ta Neither is the iminoether isolated, butsubjected to an acid alcoholysis or hydrolysis, causing cleavage of thebinding C-N and the corresponding 6- aminopenicillanic acidester havingformula VI is formed. Alternatively, the side chain may be enzymaticallycleaved in a manner known per se, e.g. such as disclosed in French Pat.No. l,576,027. In the methods generally used, the esters ofo-aminopenicillanic acid can be isolated from the reaction mixture assuch or in the form of salts with inorganic or organic acids, forinstance in the form of hydrochloride or tosylate.

Preferred compounds of the invention are:

6-( D-a-aminol'-Ethoxycarbonyloxyethyl6-(D-a-amino-m-fluorophenylacetamido)penicillanateEthoxycarbonyloxymethylo-(D-a-amino-m-fluorophenylacetamido)penicillanate l-acetoxyethyl6-(D-a-amino-p-fluorophenylacetamido)penicillanatel'-Ethoxycarbonyloxyethyl@(D-a-amino-p-fluorophenylacetamido)penicillanateEthoxycarbonyloxymethylo-(Dfl-amino-p-fluorophenylacetamido)penicillanate l -Acetoxyethylthienylacetamido)penicillanate 1 -Ethoxycarbonyloxyethylthienylacetamido)penicillanate Ethoxycarbonyloxymethylthienylacetamido)penicillanate l-acetoxy.

furylacetamido)penicillanate l-Ethoxycarbonyloxymethylfurylacetamido)penicillanate Ethoxycarbonyloxymethylfurylacetamido)penicillanate. The processes of the invention areillustrated by the following Examples:

6-(D-a-amino-3- 6-(D-a-amino-3- 6-(D-oz-amino-3- 6-(D-a-a'mino-3- 6-(D-a-amino-3- EXAMPLE 1 1'-Acetoxyethyl benzylpenicillinate a-Chloroethylacetate (312 g, 2.54 mole) was added dropwise to a stirred solution ofpotassium benzylpenicillinate (316 g, 0.85 mole) in 70 70 dioxane (1275ml) at room temperature while the mixture was kept at pH 8 by additionof sodium bicarbonate.

After the addition of the chloro ester was complete, sodium bicarbonatewas added so that the total amount of it was 413 g (5.1 mole). Thereaction mixture was stirred for 48 hours and then extracted repeatedlywith ethyl acetate. The combined extracts were washed with water andbrine, dried and evaporated in vacuo at 30 C. The oily residue wastriturated five times with toluene which was distilled off in vacuo at70 C to give the 1'-acetoxyethyl benzylpenicillinate (144 g) as an oilwhich solidified on standing. A part of it was crystallized from amixture of methanol-ether-petrolether to give the analytically purecompound m.p. 128130 C (Found: C 57.28; H 5.77; N 6.58; 23.05; S 7.82Calculated for C H N O S C 57.13 H 5.75 N 6.66 70, 0 22.83 and S 7.63The product showed strong lR-absorption at 1,785- 1,750 cm indicatingthe presence of -B-1actam rings and ester groups.

EXAMPLE 2 1'-Acetoxy-ethyl 6-aminopenicillanate hydrochloride Dryquinoline (1 1.6 g. 0.09 mole) was added at room temperature dropwise toa stirred mixture of phosphorous pentachloride (11.7 g, 0.056 mole) anddry methylene chloride (110 ml). After stirring for min the reactionmixture was chilled to C and with continued stirring treated dropwisewith l-acetoxyethyl benzylpenicillinate (20.8 g, 0.05 mole) dissolved indry methylene chloride (30 ml). 15 min after the addition was completethe mixture was chilled to 30 C and 1- propanol (37 ml) was rapidlyadded so that the temperature did not rise above 15 C. The mixture wasstirred for a further 30 min at -30 C when'ice-cooled brine (20 75 ml)was added with vigorous stirring. After stirring for 10 min at -l0 Cpetrol ether (155 ml) was added, followed by a second portion (175 ml)after'a further 10 min. yielding a crystalline precipitate which wascollected by filtration, washed with brine (70 ml) and dried to give thehydrochloride of 1-' acetoxyethyl 6-aminopenicillanate (14.2 g). Theproduct showed strong TR absorption at 1,780 1,725 cm showing thepresence of B-lactam and ester carbonyls. An analytical sample, m.p. 160C, was obtained by recrystallisation of the product from ethanol-ether.(Found: C 44:1, H 5.67, N 7.56, Calculated for C H ClN O S 42.53 H 5.37N 8.29

EXAMPLE 3 bonyloxyethyl.benaylpenicillinate (96.1 g) asa thick oil whichsolidified on standing. An analytical sample, m.p. 108 109 C, wasobtained by recrystallisation from acetone -petrol ether.

(Found: C 55.98, H 5.66, N 6.29, 0 24.34, S 7.20 7r. Calculated for C HN O S: C 56.10, H 5.83, N 6.23, 0 24.88, S 7.13 7c.)

The product showed strong IR absorption at 1,780 1,750 cm'ldue to theB-lactam and ester carbonyls.

EXAMPLE 4 EXAMPLE 5 l-Acetoxyethyl a-amino-5-methylpentylpenicillinatePotassium a-azido-5-methylpentylpenicillinate (2.9 g, 0.0071 mole) indioxane was stirred for two days with a-chloroethyl acetate (2.6 g,0.021 mole) in the presence of sodium bicarbonate (3.6 g, 0.043 mole).The reaction mixture was diluted with water and extracted with ethylacetate. The combined organic extracts were washed with water, dried andevaporated in vacuo to give 1-acetoxyethyla-azidolB-methylpentylpenicillinate (1.3 g) as an oily residue.

The product showed strong absorption at 2,120 cmand 1,750 cmcharacteristic of the azido group and the B-lactam and ester moetiesrespectively. It was dissolved in 70 ethanol and hydrogenated for 30 minover a pre-hydrogenated Raney-nickel catalyst. The, catalyst was removedby filtration and washed with ethanol. The filtrate was diluted withwater and extracted with ethyl acetate. The combined organic extractswere washed with water and then extracted with water with addition ofdilute hydrochloric acid until the pH reached 2.5. The aqueous phase wasseparated and lyophilized to give the hydrochloride of 1-acetoxyethylaamino-S-methylpentyl penicillinate (0.7 g) as slightly yellow crystals.

' The product showed strong B-lactam absorption at 1,770 1,740 cm andinhibited the growth of Staph- .aureus Oxford at 0.63 ug/ml. I

EXAMPLE 6 1-acetoxyethy1ester of 6r(D-a-azidophenylacetamiokpsa silaniseqid The sodium salt of 6-(D-a-azidophenylacetamido)- penicillanicacid (96%, 12.4 g, 0.03 mole) was dispersed in dry dimethylformamide (30ml) and under stirring and icecooling with a-chloroethylacetate (3.7 g,0.03 mole) dissolved in dry dimethylformamide' (15 ml) was added. After30 minutes the stirring was continued without cooling over the night.The reaction mixture was poured into'a saturated sodium bicarbonatesolution (500 m1), a white emulsion being obtained. This emulsion wasdiluted with H 0 (400 ml) and extracted with ether threetim'es. Thecombined ether phases were washed with H 0 and saturated sodium chloridesolution and dried. The evaporisation in vacuo of the ether solutiongave the a-acetoxyethylester of6-(D-a-azidophenylacetamido)-penicillanic acid (3.1 g) having a purityof 58 (hydroxylamine essay).

The product had a strong lR-absorption at 2,100 cm and 1,7 60 cm showingthe presence of an azido group and a B-lactame ring.

EXAMPLE 7 z y by 19- mieophea aqstanzidek penicillanate0.01-acetoxyethyl 6-(D-a-azidophenylacetamido)- penicillanate (58 7c,2.8 g), dissolved in ethyl acetate (40 ml) was treated with 0.2 M NaH PO(40 ml), acidified to pH 2.2 with 2 M hydrochloric acid and hydrogenatedover a palladium on carbon catalyst at ambient conditions for 2 hours.The caralyst was removed by filtration and washed with ethyl acetate andbuffer. The organic phase of the combined filtrates was separated andextracted with water at pH 2.1. The combined acidic aqueous phases werewashed with ether, layered with ethyl acetate and neutralized byadditions of 2 N sodium hydroxide while stirring. The aqueous phase wasseparated and extracted once with ethyl acetate. The combined ethylacetate extracts of the neutralized aqueous solution were washed withbrine and water and extracted twice with water at pH 2.5 by addition of2 M hydrochloric acid. The combined acidic aqueous extracts were washedwith ether and concentrated in vacuo at 25 C to give an oily residue,which was dissolved in isopropanol and evaporated in vacuo repeatedly togive the hydrochloride of 1'-aceto xy e thyl 6 (1);a-aminophenylacetamido)penicillanate (1.1 g) as a slightly colouredpowder with a purity of 100 70 (hydroxylamine essay).

The product inhibited the growth of Slap/1. aureus Oxford at aconcentration of 0.32 ug/ml, of E. coli and P. mirabilis at 50 ug/ml.

b.l-Acetoxyethyl 6-(D-oz-azidophenylacetamido)- penicillanate [stronglR-absorption at 2,100 cm (azido group) and 1,780 1,760 cm (B-lactam,ester)] was prepared according to the method of Example 1 from sodium6-(D-a-azidophenylacetamido)- penicillanate (397 g, 1 mole),a-chloroethyl acetate (368 g, 3 mole) and sodium bicarbonate (504 g, 6mole). The azido ester in ethylacetate (800 ml) was hydrogenated atambient conditions over a palladium "/1 on carbon catalyst g). Thecatalyst was re moved by filtration and washed with ethyl acetate. Thecombined filtrates were extracted with water at pH 2.5 by addition ofdilute hydrochloric acid. Lyophilization of the aqueous phase gave thehydrochloride of 1'- acetoxyethyl6-(D-a-aminophenylacetamido)penicillanate (110 g), m.p. 160-l64 C,(decomp.) [01],,

184C (C l,CHCl:x)l 179.9 (1 l, 50 isopropanol).

(Found: C 50.78, H 5.49, N 8.76, O 20.22, S 6.58, Cl 7.39 Calculated forC- ,H ,-,N O ;Scl: c 50.90, H 5.55, N 8.90. O 20.34, S 6.79, CI 7.51Va.)

The product showed strong IR- absorption at 1,780 1,755 cm", showing thepresence of B-lactam and ester carbonyls and was found to inhibit thegrowth of Srap/Laureus Oxford at a concentration of 0.25 ug/ml.

c.A suspension of dicyclohexylammoniuma-(onitrophenylsulphenyl)aminophenylacetate- (4.9 g, 0.01 mole) and thehydrochloride of 1'-acetoxyethyl 6-aminopenicillanate (3,4 g, 0.01 mole)in chloroform (60 ml) was stirred over night. Dicyclohexyl carbodiimide(2.2 g, 0.01 mole) was added and stirring was continued for 6 hours. Thereaction mixture was filtered and washed with water, dilute sulphuricacid, N potassium bicarbonate and water, dried and evaporated to drynessin vacuo. The yellow residue was treated with ethyl acetate, filteredand evaporated again to give a residue which was crystallized from ethylacetate/petroleum ether to give l'-acetoxyethyl 56-[D-a-(o-nitrophenylsulphenyl)aminophenylacetamido]penicil1anate (1.5g), m.p. 122-5 C.

The product had a strong IR absorption 1,780 1,750 cm showing thepresence of ,B-lactam and ester carbonyls.

The o-nitrophenylsulphurylamino ester 1.2 g, 0.002 mole) was dissolvedin 75 7c dioxane (17 ml) and adjusted to pH 3 by addition of 2 Nhydrochloric acid. Sodium iodide (1.2 g, 0.008 mole) was added and themixture was stirred and kept at pH 3 by addition of acid. After 20minutes the iodine formed was reduced with 2 N sodium thiosulphate andthe mixture was neutralized and extracted with ethyl acetate afteraddition of water. The combined organic extracts were washed with waterand extracted into water by addition of 2 N hydrochloric acid to thestirred mixture until the pH reached 2. The aqueous phase was washedwith ethyl acetate and lyophilized to give the hydrochloride of 1-acetoxyethyl 6-(D-a-aminophenylacetamido)penicilla- EXAMPLE 8 Byrepeating the procedure of Example 7a the following esters of6-(D-a-aminophenylacetamido)- penicillanic acid hydrochloride wereobtained from the corresponding azido compounds.

nate (0.15 g). The'product was identified with the one hydroxamatemethod; antibacterial activity determined by tube dilutionprocedure inbroth).

All esters of Examples 7 and 8 were hydrolyzed under the formation of6-(D-a-aminophenylacetamido)penicillanic acid by incubation (37 C) withhuman serum.

EXAMPLE 9 6-(D-a-aminophenylacetami- 1-Propiony1oxyethyl d tpsnisileuae.

Sodium 6-(D-a-azidophenylace tamido)penicillanate (6.0 g, 0.015 mole),ot-chloroethyl propionate (5.7 g,-

0.042 mole) and sodium bicarbonate (7.1 g, 0.084 mole) were stirred atroom temperature for hours in %'dioxane ml). The solid phase of thereaction mixture was removed by filtration and washed with dioxane. Thecombined filtrates were concentrated in vacuo and treated with a mixtureof benzene .(50 ml) and saturated sodium bicarbonate solution (25 ml).

The organic phase was separated, washed with brine, dried and evaporatedin vacuo to give 1- propionyloxyethyl 6-(D-a-azidophenylacetamido)-penicillanate (1.3 g) as a yellow oil, characterized by strong IRabsorption at 2,110 and 1,780 1,750 cm, showing the presence of azido,B-lactam and ester moieties.

The azido ester in ethyl acetate (50 ml) was hydrogenated for 30 minutesat ambient conditions over a prehydrogenated palladium (5 70) on carboncatalyst (1.3 g). The catalyst was removed by filtration and washed withethyl acetate and water. The pH of the combined filtrates was adjustedto 2.2 by addition of dilute hydrochloric acid. The aqueous phase wasseparated and the organic phase was extracted once more with water at pH2.2. The combined aqueous extracts were lyophilized, to give thehydrochloride of l'-propionyloxyethyl 6-(D-a-aminophenylacetamido)penicillanate (0.6 g) as an amorphous solid. I

The product showed in its 1R spectrum strong B-lactam and estercarbonyls absorption at 1,760 cm". It was found to inhibite the growthof Staph. Aureus Oxford at a concentration of 0.63 ug/ml.

EXAMPLE l0 EXAMPLE ll methoxycarbonyloxymethylesterphenylacetamido)penicillanic acid.-

By replacing the a-chloroethylacetate by monochloro-dimethylcarbonate(3.7 g, 0.03 mole) in Example 6, but otherwise operating in the same wayas in said Example the methoxycarbonyloxymethylester of6-(D-a-azidophenylacetamido)-penicillanic acid (6.6 g) having a contentof 76.5 (hydroxylamine essay) was obtained.

The product showed lR-absorption at 2,100 cm- (N -group) and 1,765 cm(B-lactame ring).

EXAMPLE 12 Methoxycarbonyloxymethyl aminophenylacetamido) penicillanate.

According to the method of Examples 11 and 8, methoxycarbonyloxymethyl6-(D-a-aminophcnylacetamido)penicillanatc was prepared, starting fromsodium 6-(D-a-azidophenylacetamido) penicillanate (24.2g, 0.06 mole),chloromcthyl methylcarbonatc (6.2 g, 0.06 mole). The compound wasisolated as its hydrochloride (5.6 g) m.p. 145-l50C(decomp.) [04],,+1825 (C 1, CHCla); 191.2 (C =1, 50

of 6-(D-a-azidoisopropanol), (Found C 48.04, H 5.27, O 23.67, N 8.94, S6.57, and Cl 7.36 76. Calculated for C, H ,N O S Cl C 48.15, H 5.10. N8.87, O 23.63, S 6.77 and Cl 7.48 7(). The product showed strong IRabsorption at 1,780 1,760 cmshowing the presence of B-lactam and estercarbonyls. It was found to inhibit the growth of Staph- .Aureus oxfordat a concentration of 0.13 ug/ml EXAMPLE l3 l'-ethoxycarbonyloxy-ethylphenylacetamido)penicillanate 1 i The sodium salt of 6-(D-a-azidophenylacetamido)- penicillanic acid (4.0 g 0.01 mole) wasdispersed in dry dimethyl formamide (10 ml) and treated under stirringat 50C with a-chloro-diethyl carbonate (1.5 g 0.01 mole) dissolved indry dimethyl formamide (6 ml). After stirring for 20 hours at 50 C thereaction mixture was cooled and poured into an icecold, saturated sodiumbicarbonat solution and extracted three times with ether. The combinedether phases were washed with water and brine and dried. Evaporation invacuum gave the a-ethyoxycarbonyloxi ethylester of6-(D-aazidophenylacetamido)-penicillanic acid (2.6 g) in the form of ayellow-brown glass.

The product had strong lR-absorption at 2,100 cm and 1,750 cm showingthe presence of an azido group and B-lactame ring and an ester group.

EXAMPLE l4 l-ethoxycarbonyloxy-ethyl phenylacetamido) penicillanate Thea-ethoxycarbonyloxy ethylester of-(D-a-azidophenylacetamido)-penicillanic acid (2.5 g) was dissolved inethyl acetate (40 ml) and 0.4 mole NaH PO, acidified to pH 2.2 with 2mole hydrochloric acid (40 ml) and 10 palladium on carbon catalyst (4 g)was added and hydrogenation was carried out at normal pressure for 0.5hour. The catalyst was separated by filtration and washed with ethylacetate and buffer solution. The filtrate was separated and the organicphase was extracted with water at pH 2.1. The combined acid water phaseswere washed with ether, ethyl acetate was added and neutralisation wascarried out under stirring with 2 mole NaOH. The phases were separatedand the 6-(D-a-aminowater solution was reextracted with ethyl acetate.The

two ethyl acetate extracts were'combined, washed with brine and waterand extracted with water at pH 2.5 by the addition of 2 molehydrochloric acid. The extraction of the ethylacetate phase wasrepreated once. The combined acid water extracts were washed with etherand concentrated in vacuo at 25 C. The semi-solid residue was dissolvedin isopropanole and reconcentrated, the procedure being repeated once.The residue was stirred with ether, filtrated and-washed with ether,giving the a-ethoxycarbonyloxy ethylester of 6-(D-aaminophenylacetamido) penicillanicacid hydrochloride (0.8 g) as a yellow-whitepowder having a content of 86.4 (hydroxylamine essay).

The product inhibited the growth of .S'luphAureus Oxford at aconcentration of 0.13 ug/ml and of E. coli a! ,ug/ml (solution test onagar) and contained inits lR-spectrum a strong absorption band at 1,750cm" formamide (50 ml) with oz-azidophenylacetyl chloride (2 g, 0.01mole) in presence of triethylamine (2 g, 0.01 mole). After dilution ofthe reaction mixture with water the ester was isolated by extractionwith ethyl acetate.

EXAMPLE 15 1-Ethoxycarbonyloxyethyl phenylacetamido)penicillanateAccording to the method of Example 1 l-ethoxycarbonyloxyethyl6-(D-a-azidophenylacetamido)penicillanate (98 g) was prepared fromsodium 6-(D-a-azidophenylacetamido)penicillanate (397 g, 1 mole),a-chlorodiethylcarbonate (458 g, 3 mole) and sodium bicarbonate (504 g,6 mole), The product showed strong IR absorption at 2,090 cm and 1,7801,750 cm showing the presence of azido group and B-lactam and estercarbonyls.

lt was dissolved in ethyl acetate (700 ml) and hydrogenated at ambientconditions over a palladium (5 on carbon catalyst 18 g). The catalystwas removed by filtration and washed with ethyl acetate. The combinedfiltrates were extracted with water at pH 2.5 by addition of dilutehydrochloric acid. Lyophilization of the aqueous phase gave thehydrochloride of l-ethoxycarbonyloxyethyl6-(D-aaminophenylacetamido)penicillante (94 g), m.p. 171 176 C (decomp)+16l.5 (C l, CHCl +l7l.8 (C l, 50% isopropanol) (Found C 50.06, H 5.82,O 22.26, N 8.35. S 6.56, Cl 7.19 70. Calculated for C ,H. ,,,N;,O,SCI C50.24, H 5.68, N 8.37, O 22.31, S 6.39,C17.06 /v.)

The product showed strong IR absorption at 1,780 1,750 cmdue to theB-lactam and ester carbonyls, and was found to inhibit the growth ofSMPILALHGLIS Oxford at at concentrations of 0.13 ug/ml.

EXAMPLE 16 l'-Ethoxycarbonyloxyethyl phenylacetamido)penicillanate a. Toa suspension of the hydrochloride of a-aminophenylacetyl chloride (2.6g, 0.0125 mole) stirred in dry chloroform (50 ml) under dry nitrogensodium bicarbonate (1.1 g, 0.0125 mole) and the hydrochloride ofl'-ethoxycarbonyloxyethyl 6-aminopenicillanate (3.3 g, 0.01 mole) wasadded. After stirring for 90 minutes the mixture was filtered andisopropanol ml) was added to the filtrate. Evaporation in vacuo at roomtemperature gave an oily residue which on dilution with petrol etherprecipitated the hydrochloride of l-ethoxycarbonyloxyethyl6-(D-a-aminophenylacetamido)- penicillanate (1.5 g). The product waspurified by precipitation from acetone-petrolether.

The identity of the product was ascertained by comparison ofits IRspectrum with that of the product from Example 15.

[2. sodium N-( l-methoxycarbonyl-propen-Z-yl)-aaminophenylacctate (2.5g, 0.0092 mole) and N methylmorpholine (0.05 ml) in dry ethyl acetate(40 ml) were stirred and treated at l5 C with isobutyl chloroformate(1.4 g, 0.01 mole). After 6 min l'-ethoxycarbonyloxyethyl6-aminopenicillanate (0.01 mole) in ethyl acetate (20 ml) was addeddropwise while stirring was continued and the temperature was maintainedat 15 C. 10 min after the addition was complete the cooling bath wasremoved and stirring was continued for 45 min. The reaction mixture waswashed with water, 0.5 mole sodium bicarbonate and 6-(D-a-amino-Propoxycarbonyloxymethyl water again, dried and concentrated in vacuo toan oily residue which was treated with tetrhydrofurane (20 ml) and water(20 ml). The mixture was acidified to pH 2.5 by addition of 2 Nhydrochloric acid. After 30 min most of the tetrahydrofurane was removedin vacuo and the acidic aqueous phase was washed with ethyl acetate andevaporated to give the hydrochloride of 1-ethoxycarbonyloxyethyl 6-(D-a-aminophenylacetamido)penicillanate (0.35 g) as an amorphous solid,identified as the product obtained above by means of its IR spectrum.

The solution of 1'-ethoxycarbonyloxyethyl 6- aminopenicillanate used wasobtained by neutralization of an aqueous solution of its hydrochlorideand extraction with ethyl acetate.

EXAMPLE 17 Ethoxycarbonyloxymethyl phenylacetamido)penicillanate Astirred suspension of sodium 6-(D-aazidophenylacetamido) penicillanate(12.1 g, 0.03 mole) in dry dimethylformamide (30 ml) was treated withchloromethylethyl carbonate (4.2 g, 0.03 mole) in dry dimethylformamide(15 ml) in the ice-bath. After 45 min the cooling bath was removed andstirring was continued over night. The dark reaction mixture was pouredinto twice its volume of saturated sodium bicarbonate solution and thesolid phase found was removed by filtration and washed with water andether. The combined filtrates were extracted with ether. The etherextracts were washed with water, dried and evaporated in vacuo, at roomtemperature to give ethoxycarbonyloxymethyl 6-( D-a-aminophenylacetamidopenicillanatie, (4.7 g) as a brownish oil. The product showed strongabsorption bands in IR at 2,120 cm" and 1,780 50 cm attributable to theazido group and the B-lactam and ester carbonyls respectively.

The azido ester (2.6 g) was dissolved in 60 ethanol (50 ml) andhydrogenated for 30 min at 60 p.s.i. over a prehydrogenated Raney-nickelcatalyst. The catalyst was removed by filtration and washed withethanol. The filtrate was diluted with water and extracted with ethylacetate. The combined organic exstracts were washed with water and thenextracted with water by addition of dilute hydrochloric acid until thepH reacted 2.5. The aqueous phase was separated and lyophilized to givethe crystalline hydrochloride of l'-ethoxycarbonyloxymethyl6-(D-oz-aminophenylacetamido)- penicillanate (0.7 g). The product showedstrong IR absorption at 1,760 cm and inhbited the growth ofStaplz.Aureus Oxford at 0.05 ug/ml.

EXAMPLE l8 6-(D-a-amino- 6-(D-aaminophenylacetamido) penicillanateAccording to the procedure of Example 17 propoxycarbonyloxymethyl6-(D-a-azidophenylacetamido)- penicillanate (5.2 g) was obtained fromsodium 6-(D- a-azidophenylacetamido)penicillanate (12.1 g, 0.03 mole)and chloromethyl n-propyl carbonate (4.6 g, 0.03 mole). lt exhibitedstrong lR-absorption at 2,120 cm and 1,780 1,740 cmshowing the presenceof azido group and of B-lactam and ester carbonyls respectively.

On hydrogenation over Raney-nickel the azido ester (2.9 g) gavepropoxycarbonyloxymethyl6-(D-aaminophenylacetamido)penicillanateisolated as its hydrochloride1.0 g). It showed strong B-lactam and ester absorption bands in IR at1,775 cm, and inhibited the growth of Staph. Aureus Oxford at aconcentration of 0.05 ug/ml.

EXAMPLE l9 lsopropoxycarbonyloxymethyl phenylacetamido)penicillanate 1nthe manner described in Example 17 isopropoxycarbonyloxymethyl6-(D-oz-azidophenylacetamido)- penicillanate (7.2 g) was obtained fromsodium 6-(D- a-azidophenylacetamido)penicillanate (9.9 g, 0.025 mole)and chloromethyl isopropyl carbonate (3.8 g, 0.025 mole). lts IRspectrum contained strong absorption bands at 2,110 cm (azido group) and1,780 1,740 cm (B-lactam and ester carbonyls). v

Hydrogenation over Raney-nickel converted the azido ester (4.4 g) intoisopropoxycarbonyloxymethyl 6-(D-a'aminophenylacetamido)penicillanateisolated as its hydrochloride (1.3 g). lt showed strong absorption in IRat 1.760 cm characteristic for B-lactam and ester carbonyls andinhibited the growth of Staph. Aureus Oxford at a concentration of 0.05ag/ml.

EXAMPLE 20 Butoxycarbonyloxymethyl phenylacetamido)penicillanateAccording to the procedure of Example 17 butoxycarbonyloxymethyl6-(D-a-azidophenylacetamido)- penicillanate (6.0 g) was prepared fromsodium 6-(D- a-azidophenylacetamido)penicillanate (9.9 g, 0.025 mole)and chloromethyl-n-butyl carbonate (4.2 g, 0.025 mole). The compoundshowed strong 1R absorption at 2,120 cm (azido group) and 1,780 1,750 cm(B-lactam and ester carbonyls).

The azido ester (2.9 g) was hydrogenated over Raney-nickel to givebutoxycarbonyloxymethyl 6-(D- a-aminophenylacetamido) penicillanateisolated as its hydrochloride (1.0 g). It showed strong IR absorption at1,780 1,770 cm" (B-lactam and ester carbonyls) and inhibited the growthof Staph. Aureus Oxford at a concentration of 0.05 ag/ml.

EXAMPLE 21 Ethylcarbamatemethylester of phenylacetamido)-penicillanicacid To an agitated and icecooled dispersion of the sodium salt of6-(D-a-azidophenylacetamido)- penicillanic acid (98 7c, 7.2 g, 0.018mole) in dimethylformamide 18 ml) N-chloromethyl ethyluretan (2.1 g,0.018 mole) dissolved in dimethylformamide (10 ml) was added. After 40minutes the stirring was continued over night without cooling.

The reaction mixture was poured into cold sodium bicarbonate solution(400 ml) and extracted twice with ether. The combined ether phases werewashed with water and sodium chloride solution and dried. Evaporation ofthe ether extract gave the ethylcarbamatemethylester of6-(D-a-azidophenylacetamido)- penicillanic acid (2.6 g).

After reprecipitation of the product from methylene chloride/petroleumether 2.3 g was obtained having a purity of 57.5 71.

6-( D-a-aminoo-(D-a-amino- EXA M PLE 22 l'-acetoxy-ethyl 6-(D-a-amino-m-fluoro phenylacetamido )penicillanate Hydrochloride of l-acetoxyethyl 6- aminopenicillanate (3.4 g, 0.01 mole) in drydimethylformamide (50 ml) was stirred in the ice-bath and treated withN,N-dimethylaniline (2,4 g, 0.02 mole) followed by a solution ofa-azido-m-fluorophenylacetyl chloride (2.1 g, 0.01 mole) in dry ether (5ml). After stirring for 1 hour water (100 ml) was added and the mixturewas adjusted to pH 6.5 and extracted three times with ethyl acetate. Thecombined organic extracts wre washed with water and brine. dried andevaporated in vacuo to give l-acetoxyethyl6-(D-aazido-m-fluorophenylacetamido)penicillanate (1.5 g) as an oilyresidue. This product showed strong 1R absorption at 2,1 10 and 1,7801,750 cm indicating the presence of the azido group and B-lactam andester carbonyls.

The azido ester (0.7 g) was dissolved in 60 72 ethanol (7 ml) and addedto prehydrogenated Raney nickel catalyst (0.7 g) in 80 ethanol (5 ml)and hydrogenated for 30 min at ambient conditions. The catalyst wasremoved by filtration and washed with ethanol. The combined filtrateswere concentrated in vacuo at 35 C to an oily residue which wasdissolved in ethyl acetate. Water (20 ml) was added and the pH wasadjusted to 1.6 by addition of2 N hydrochloric acid to the stirredmixture. The aqueous phase was separated and evaporated in vacuo at 35 Cto give the hydrochloride of 1 '-acetoxyethyl 6-(D-a-amino-m-fluorophenylacetamido)penicillanate (0.4 g) as a crystallineresidue.

The product was found to inhibit the growth of Staplr.Aureus Oxford at aconcentration of 0.25 ug/ml and to contain in its IR spectrum a strongB-lactam absorption at 1,760 cm.

EXAMPLE 23 1-Ethyloxycarbonyloxyethyl6-(D-a-amino-m-fluorophenylacetamido)penicillanate To sodium6-(D-a-azido-m-fluorophenylacetamido)- penicillanate (4.2 g, 0.01 mole)and sodium bicarbonate (5.1 g, 0.06 mole) in dioxane (15 ml)a-chloro-diethylcarbonate (4.6 g, 0.03 mole) was added and the mixturewas stirred for two days at room temperature. The reaction mixture wasfiltered, the residue washed with dioxane and the combined filtrateswere evaporated to dryness in vacuo. The residue was dissolved in amixture of benzene (40 ml) and saturated aqueous sodium bicarbonate (20ml). The organic phase was separated, washed with brine, dried andevaporated to give 1-ethyloxycarbonyloxyethyl 6-(D-a-azido-m-fluorophenylacetamido)penicillanate 1.0

The product showed strong IR absorption at 2,100 and 1,750 cm"corresponding to the azido group and the B-lactam and ester moieties,respectively.

It was dissolved in ethylacetate (10 ml) and added to palladium oncarbon (5%) hydrogenation catalyst (0.5 g), prehydrogenated inethylacetate (5 ml) and hydrogenated for 2 hours under ambientconditions. The catalyst was filtered off and washed with ethylacetate.

The product was found to contain in its IR spectrum a strong absorptionat 1,780 cm corresponding to a B-lactam ring. 1t inhibited the growth ofStaph. Aureus Oxford at a concentration of 0.13 tLg/mole.

EXAMPLE 24 1'-Acetoxyethyl 6-(D-a-amino-p-fluorophenylacetamide)penicillanate Using the proceduredescribed in Example 23 1'- acetoxyethyl 6(D-a-azido-p-fluorophenylacetamido)- penicillanate (2.2 g) was preparedfrom sodium 6-(D- a-azido-p-fluorophenylacetamido) penicillanate (4.2g,-

0.01 mole), a-chloroethyl acetate (3.7 g, 0.03 mole) and sodiumbicarbonate (5 g, 0.06 mole). The IR spectrum of the product showedstrong absorptions at 2,1 cm" (azido group) and 1,775 1,750 cm (B-lactamand ester carbonyls).

The azido ester was hydrogenated according to the procedure described inExample 22 over Raney-nickel catalyst to give the hydrochloride ofl'-acetoxyethyl 6-(D-a-amino-p-fluorophenylacetamido)penicillanate (1.5g) as slightly yellow crystals. The product showed in its IR spectrum astrong Blactum and ester absorption at 1,765 cm and was found to inhibitthe growth of Staph. Aureus Oxford at a concentration of 0.25 pig/ml.

EXAMPLE 25 l'-Ethyloxycarbonyloxyethyl phenylacetamido)penicillanate Tosodium 6-(D-oz-azido-p-fluorophenylacetamido)- penicillanate (4.2 g,0.01 mole) and sodium bicarbonate (5.1 g, 0.06 mole) in 70 "/1 dioxaneml) a-chlorodiethylcarbonate (4,6 g, 0.03 mole) was added and themixture was stirred for 2 days at room temperature. The reaction mixturewas filtered; the residue washed with dioxane and the combined filtrateswere evaporated to dryness in vacuo. The residue was dissolved in amixture of benzene (40 ml) and saturated aqueous sodium bicarbonate ml).The organic phase was separated, washed with brine, dried and evaporatedin vacuo to give l-ethyloxycarbonyloxyethyl6-(D-a-azido-p-fluorophenylacetamido)penicillanate (1.9 g).

The product showed strong IR absorption at 2,110 and 1,750 emcorresponding to the azido group and the B-lactam and ester moieties,respectively. It was dissolved in ethyl acetate 10 ml) and added toprehydrogenated palladium (5 7c) as carbon catalyst (0.5 g) in ethylacetate (5 ml) and hydrogenated for 2 hours under ambient conditions.The catalyst was removed by filtration and washed with ethyl acetate.Water (10 ml) was added to the combined filtrates and pH was adjusted to2.2 by addition of 2 N hydrochloric acid to the stirred mixture. Theaqueous phase was separated,

washed with ether and evaporated to dryness in vacuo EXAMPLE 26l'-Acetoxyetyl 6-(0t-amino-p-chlorophenylacetamido)penicillanate and1,770 1,750 cm corresponding to the azido group and the B-lactam andester moieties, respectively. It was dissolved in 70 70 ethanol (7 ml)and hydrogenated for 30 min at 60 p.s.i. over a prehydrogenatedRaney-nickel catalyst. The catalyst was filtered off and washed withethanol. The combined filtrates were diluted with water and extractedwith ethyl acetate. Water was added to the organic phase and the pH ofthe mixture was adjusted to pH-2.2 by addition of dilute hydrochloricacid. Lyophilization of the aqueous phase gave 1 '-acetoxyethyl 6-(a-amino-pchlorophenylacetamido) penicillanate hydrochloride (0.23 g) asa crystalline residue.

The product was found to contain a strong B-lactam absorption in its IRspectrum at 1,760 1,740 cm and to inhibit the growth of Staph. AureusOxford at a concentration of 0.63 ug/ml.

EXAMPLE 27 1-Acetoxyethyl6-(D-a-amino-p-hydroxyphenylacetamido)penicillinate SodiumN-(1methoxyearbonylpropen-2-yl)-aamino-p-hydroxyphenyl acetate (2.9 g,0.01 mole) in dry ethyl acetate (40 ml) was stirred and treated at l5 to'20 C with N-methylmorpholine (a few drops) followed byisobutylchloroformate 1.4 g, 0.01

' mole). After 5 min a solution of l-acetoxyethyl 6- aminopenicillanate(3 g, 0.01 mole) in ethyl acetate (20 ml) chilled to l5C was rapidlyadded. After stirring for 60 min without external cooling the reactionmixture was washed with water, 0.5 M potassium bicarbonate, and water,dried and evaporated in vacuo at room temperature to dryness. Theresidue was dissolved in a 1:1 mixture of tetrahydrofurane and water (50ml), stirred and kept for 30 min at pH 2.5 by addition of 2 Mhydrochloric acid. The tetrahydrofurane was evaporated in vacuo and theremaining aqueous phase was washed with ethyl acetate, diluted withisopropanol and evaporated in vacuo at room temperature to give thehydrochloride of 1'-acetoxyethyl6-(D-otamino-p-hydroxyphenylacetamido)penicillanate (2.3 g) as whitecrystals. The product showed in 1R strong absorption bands at 1,760 cmattributable to the B-lactam and ester carbonyls. It was found toinhibit the growth of Staph. Aureus Oxford at a concentration of 0.25ttg/ml.

The solution I of l-ac etoxyethyl 6-(D-aaminophenylacetamido)penicillanate used was prepared by neutralization of an aqueous solutionofits hydrochloride and extraction with ethyl acetate.

EXAMPLE 28 1'-Ethoxycarbonyloxyethyl phenylacetamido )penicillanateAccording to the procedure of Example 27 l-ethox-6-(D-o1-amino-p-hydroxyycarbonyloxyethyl 6-(D-a-amino-p-hydroxyphenylacetamido)penicillanate isolated as its hydrochloride (1.4 g) wasprepared from sodium N-(lmethoxycarbonylpropen-2-yl)-a-amino-phydroxyphenyl acetate (2.9 g, 0.01mole) and l'-ethoxycarbonyloxyethyl 6-aminopenicillanate (0.01 mole).The product contained in its IR spectrum strong absorption at 1,760 cm,showing the presence of B-lactam and ester carbonyls. it was found toinhibit the growth of Staph. Aureus Oxford at 0.25 pg/ml.

EXAMPLE 29 and the temperature was mainteined at l5 C. 10 min after theaddition was complete the cooling bath was removed and stirring wascontinued for 45 min. The reaction mixture was washed withwater, 0.5mole sodium bicarbonate and water again, dried and concentratcd in vacuoto an oily residue which was treated with tetrahydrofurane ml) and water(20 ml) and kept at pH 2.5 for min by addition of dilute hydrochloricacid. Most of the tetrahydrofurane was evaporated in vacuo and theaqueous phase was washed with ethyl acetate and lyophilized to give thehydrochloride of l l -acetoxyethyl 6-(a-amino-3-furylacetamido)penicillanate (1.9 g) as a white glassy mass.

The product was found to inhibit the growth of Staph. Aureus Oxford at aconcentration of 0.63 ug/ml and to contain in its IR spectrum a strongabsorption at 1,760 cm attributable to the B-lactam ring and the estergroups.

The solution of l-acetoxyethyl 6-aminopenicillanate was obtained byneutralization and extraction with ethyl acetate of an aqueous solutionof its hydrochloride. The sodium N-( l-methoxycarbonylpropen-Z-yl)-a-amino-3-furyl acetate was prepared by reaction of sodiuma-amino-3-furyl acetate and methyl acetoacetate in refluxing toluene.

strong absorption at 1760 cm" in its IR spectrum, showing the presenceof B-lactam and ester moieties.

EXAMPLE 31 l-Acetoxyethyl 6-(oz-amino-3- thienylacetamido)penicillanateThe hydrochloric of l-acetoxyethyl 6-(a amino-3-thienylacetamido)penicillanate (1.2 g) was obtained according to theprocedure of- Example 29 from sodium N-(l-methoxycarbonylpropen-Z-yl)-a-amino-3-thienyl acetate (28 g, 0.01mole) and l'-acetox vethyl 6- aminopenicillanate (0.01 mole).

The product showed in its IR spectrum strong fi-lactam and esterabsorption at 1,760 cm.

The sodium N-(1-methoxycarbonylpropen-Z-yl)-aamino-3-thienyl acetate wasobtained by reaction of methylacetoacetate and sodium a-amino-3-thienylacetate in refluxing toluene.

EXAMPLE 32 Oral absorption in' humans of l-acetoxyethyl andl-ethoxycarbonyloxyethyl 6-( D-a-aminophenylacetamido)penicillanate Theoral absorption of l-ethoxycarbonyloxyethyl 6-(D-a-aminophenylacetamino)penicillanate (l) and of l-acetoxyethyl 6-(D-a-.

aminophenylacetamido)penicillante (ll) was studied in a cross-overexperiment in nine healthy male volunteers using 6-(D-a-aminophenylacetamido) penicillanic acid (ampicillin) as a reference.The compounds were administered in tablets with the followingcompos1t10n:

I ll

Penicillin ester 359 mg 338 mg 1 Corn starch 101. mg 102 mg Magnesiumstearate 10 mg 10 mg The amounts of the esters correspond to 250 mg offree ampieillin. The reference compound was given in a commerciallyavailable tablet (Doktacillin 0.25 g) containing 250 mg of freeampieillin. The tablets were taken on an empty stomach. Blood sampleswere drawn at preset intervals and analyzed for their contents ofampicillin using a microbiological essay. The following mean serumlevels (in tg/ml) with standard error of the mean were found;

Time (hours) C0111 pound 1 5.99 4.79 3.47 2.51 1.14 0.71 0.20 0.06910.90 10.35 10.32 10.16 10.13 10.10 10.026 10.011 11 7.48 5.38 3.88 2.801.17 0.68 0.19 0.065 10.86 10.38 10.33 10.38 10.18 v 10.11 10.035 @016Ampi- 0.48 1.49 2.30 2.28 1.66 1.52 0.35 0.12

cillin 10.17 10.29 10.32 10.31 10.25 10.25 10.062 10.026

EXAMPLE 30 The mean cumulative urinary excretions after 8 hours inpercentage of the dose given were: l-Ethoxycarbonyloxyethyl 6-( 60-am1no-3- thienylacetamido)penicillanate V In the manner described inExample 29 the hydrochloride of 1'-ethoxycarbonyloxyethyl 6-(a-amino-3-thienylacetamido)penicillanate was prepared froml-ethoxycarbonyloxyethyl 6-aminopenicillanate and sodium N-(l-methoxy-carbonylpropen-2-yl)-a-amino- 3-thienyl acetate. The productwas found to contain a 53.4 53.7 Ampieillin 34.5

g, EXAMPLE 33 Hydrolysis of aminopenicillin esters into thecorresponding aminopenicillins in buffer and in presence of human serumThe hydrolysis of the esters of the aminopenicillins prepared accordingto the invention were determined in the same buffer containing l humanserum. The test solutions were extracted with ethyl acetate to removeunhydroly'zed ester and were analyzed microbiologically for theircontents of aminopenicillin. Blank i b ff l ti with and witho t hu annnserum values were obtained by dissolving the esters in phosbeingpresent. The esters were i b t d f 30 i t phate buffer at the sameconcentration and immedi- 37 C at a concentration f 1 i sorensens atelyperform the extraction. The values in the table are phosphate buffer (pH7.4) and in another experiment Corrected for The blank Valuess ("lluR-ClI-C 0-011-011-01! Pm NH: ooN- ou-ooott iilil lll lnlll.li"5Y1 Z.

Phosphate u l R Br riifhlii illr? 13,1131

(CH3)2CHCH2CH2 CH3 5 6. 0 15. 3

-('JH-OCOCH3 C 115 Same asabove 7 7. 0 17.0

CaHs CH: 9 6.8 I 17.0

Jan-00002115 (1H5 1 CH3 10 5.2 12.1 -C 3H-OCOCH(OHa)2 CaHa CH3 10 I 4. 914. 3

C0115 431-1200000113 a 24, 8 77. u

can cm 13 s 8 s7 1 -(JHOCOOOzlh Calls -CI[2OCOOCE"5 17 13. s m. 7 0cmCII2OCOOU3H7 1x 141' m. 7 (1,715 -o'1i-. o00o0n((.:1It)! in 15. 2 (as. 4Sill? EHZiGiiiiblla L 3'5 1%.; m-Fc61Ir CH3 22 3. 1 1s. 4

JH-0coom m-F-CJL CH3 23 I 2.0 37.7

-( JHOCOOC2Ha p-F-CaH; I CH: 24 J 14.3

-oH-oco0m p-F-CfiH4 CH3 25 1 4 2o. 2

- 3H0o00Q2H5 p-C1CaH4 CH3 26 10. 8 15. 6 A an-0000113 p-HO-CsH4 Same asabove 27 2. 5 I 6. 8

p-HO-CgHr CH: 28 5. 0 33. 1

CHOCO0OzHs CH3 20 3.5 12.4 H J 3H-OCOCHa \o I 7 U- Same as above 30 6.78. 0 U

Same as above e. CH3 31 7.0 I 35.2

EXAMPLE 34 Pharmaceutical formulations For preparation of tablets thefollowing compositions were made:

a) I'-Acctoxyethyl o(D-uaminophcnylacetamido) penicillanatehydrochloride 338 mg Starch I mg Magnesium stearate mg b)l-Ethoxycarlmnyloxyethyl 6-( D-u-aminophenylacetamido)pcnicillanatchydrochloride 359 mg Starch 100 mg Magnesium stearatc l0 mg c)Ethoxyearhonyloxyn'icthyl o-(D-u-aminophcnylacetamido)penicillanatchydrochloride 350 mg Calcium carbonate 100 mg Magnesium stearate 10 mgd) l'-Ethoxycarbonyloxymethyl 6-(D-a-aminophenylacctamido)penicillanatehydrochloride 359 mg Lactose 100 mg Magnesium stearate l0 mg e)l'-Ethoxycarbonyloxymethyl(w-(D-a-amino-mfluorophcnylacetamido)penicillanate 370 mg hydrochlorideMicrocrystalline cellulose (Avicel) 100 mg Magnesium stcarute l0 mg l)l-Acetoxyethyl 6-(D-(xaminophcnylacetamido) penicillanate hydrochloride338 mg Calcium carbonate 100 mg Lactose 100 mg Magnesium stearate l0 mgFor filling in capsules the following formulations were made: g)l'-aceto.\yethyl 6-(D-oz-aminophenylacetamido) penicillanatehydrochloride 350 mg Magnesium stearate 5 mg h)l'-Ethoxycarbonyloxyethyl 6-(D-a aminophenylacetamidolpenicillanatehydrochloride 350 mg Lactose 40 mg 5 mg Magnesium stearate For oralsuspensions the following formulations were prepared:

i) l '-Acetoxyethyl 6-(D-a-aminophcnylacetamido) sorbitol What isclaimed is: l. A compound of the structural formula and pharmaceuticallyacceptable acid addition salts thereof in which formula R is the radicalwherein R is selected from the group consisting of hydrogen and hydroxyand R is selected from the group consisting of hydrogen and halogen,such as chlorine and fluorine, and wherein R is selected from the groupconsisting of in which formulas R is selected from the group consistingof alkyl groups containing one to four carbon atoms; and'R is selectedfrom the group consisting of H and CH;,.

2. A compound according to claim 1 having the structural formula andpharmaceutically acceptable acid addition salts thereof.

3. A compound according to claim 1 having the structural formulao-dihocooonr and pharmaceutically acceptableacid addition salts thereof.4

4. A compound according to claim 1 having the structural formula 6. Acompound according-to claim 1 having the structural formula andPharmaceutically acceptable acid addition Saltsand pharmaceuticallyacceptable acid addition salts thereof. thereof.

A mp accordmg to Clalm 1 havlng tha 11. A compound according to claim 1having the structural formula tructural formula /s\ /CII3 S /C a 0()ll-(1()NlI(Ilfl)II (|?()Ila F(|3HCONH(|JH(JH (It-cm r m: 0:=(1N-UII(|,| NH O=C -N CH.

O "W V cm OOH OCOOC Hr I -OOHOCOCH1 and pharmaceutically acceptable acidaddition salts thereof.

8. A compound according to claim 1 having the structural formula andpharmaceutically acceptable acid addition salts thereof.

12. A compound according to claim 1 having the structural formula andpharmaceutically acceptable acid addition salts 3 thereof.

9. A compound according to claim 1 having the structural formula I S OH;

OCH OCOOC'JH 2 5 and pharmaceutically acceptable acid addition salts 0-(JH-OCOOCgB5 thereof.

14. A compound according to claim 1 wherein R is and pharmaceuticallyacceptable acid addition salts thereof. CH5

10. A compound according to claim 1 having the T E structural formulaUNITED STATES PATENT AND TRADEMARK OFFICE QETIFICATE OF CORRECTEUNPATENT NO. 3,873,521 DATED March 25, 1975 INVENTOR(S) Bertil Ake Ekstromet a1.

it is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Abstract, "ll 0 should be 111 0 H II CI-I- O-"C"-R"'" CH--O- C-O--R"First page, Item [73] "Lakemedal" should be Lakemedel Col, 5, line 5 S 9"disceases" should be diseases Cols i and 1, in the table, under heading"R", in Example 22,

delete "C HS"; and 4, in the table, under heading "Example", oppositeExamples 21 and 8 delete same line, delete "8";

line next below the foregoing, under the column I LN "Example", insert 8Col. 5, last line "CH" should be CH (Fol. 6 line 60, embodiments" shouldbe embodiment C 6l Z, line 77, "characteristica" should becharacteristics- Co l 7 line 3?, "bensyl. should be benzyl EST. 7, line5, "tripehnylmethyl" should be triphenylmethyl (E. 5, line H, "they"should be that Col 8, line 18, "khown" should be known C o l line 2 F,"elavated" should be elevated (3 51 5, line El, "Nickel" should benickel (1 5? line 57) "hexametylentet" should be hexamethylentet m 5,line 3?, after "as", insert a C ol line H, "ease" should be case 61 TOclose the spacing between the following groups line "ethyl" and "6-(Dor-amino";

line 32, between "ethyl" and"6-(Daamino";

line 3 between "methyl" and "6- (Db-amino"; line i5 between "ethyl" and"6- (D-or-amino"; line 37?, between "ethyl" and "6- (Db-amino"; line 4 0between "methyl" and "6- (D-wamino"; line 4 2, between "ethyl" and "6-(DD-amino";

UNITED STATES PATENT AND TRADEMARK OFFICE EEHECATE I CORRECTION PATENTNO. 3,873,521

DATED March 25, 1975 INV ENTOR(S) Bertil Ake Ekstrom et al It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

C o l. E, line ill, between "ethyl" and "6- (D-cx-amino"; line i,between "ethyl and "6 (D-o-amin0"; line 57, between "ethyl" and"6-(D-wamino"; line E, between "methyl" and "6- (DD-amino"; line @l,between "acetoxy" and "6- (Daamino";

line 3, between "methyl" and "6(Ddamin0"; and line 5 5, between "methyl"and "6- (Do-amino";

ti l 10, betweQq lines 35 a 36 41 6% 42 47 G 48 53 8 54 and 60 8, 61,insert (at the left-hand margin) Col l2, line 61, "l'acetoxy should bel'acetoxyethyl 9 l l l line 4 8 "TR" should be IR Col 12, line 29,"dazido 16" should be oLazido-6-; (T5 TIY, Example 7 close the spacingbetween "ethyl" and w "6 (D-o-amino"; Col 13, line 13, "catalyst" shouldbe catalyst s E E, line E, "additions" should be addition Co 13, line 5l "Scl" should be SCl C o 1?, line 6 before ".(4 9", delete the hyphen;E T T, lixamp 2, close the spacing between "ethyl" and w "6- (DD-amino";01 15, Example 12 clcse the spacing between "methyl" and vv6 D OLH; Q.15, line ;Z 2, "inhibite" should be inhibit Co T3, line 28 "Example 17''should be Example 9 F T5, line F5, "methoxy. should be Methoxy. E5 fi,ExampTe l3 close the spacing between "ethyl" an "6- (D-d-amino"; Q 9 ll2, Example H, close the spacing between "ethyl" and "6-(D-cx-amin0";Col. 16, line g, "oxford" should be Oxford UNITED sTATEs PATENT ANDTRADEMARK oEEIoE' fiERTIFlCATE 0F COREECTIQN PATENTNO.I 3,873,521

DATED March 25, 1975 l y oms) Bertil Ake Ekstrom et al it is certifiedthat error appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Col a lg, line 2 "bicarbonat" should be bicarbonate Col E, line 3;,"ethyoxycarbonyloxi" should be ethyoxycarbonyloxy line 50, "repreated"should be repeated Example l5, close the spacing between "ethyl" and "6-(Daamino";

Col C Col l7 Example 16, close the spacing between "ethyl" and "6-(Ddamino"; Qgl. H, line Q, "penicillante" should be pen icillanate E. 17line 2 7, after (decomp. insert [on] o l. E, line 5Q, "sodium" should beSodium Co l 8 Example u, close the spacing between "methyl" and l E,Example E, close the spacing between "methyl" and n vv; Col. 18, line2,l"tetrhydrofurane" should be tetrahydrofurane; o l 13?, line B,"exstracts" should be extracts o6 l line E, "reacted" should be reachedE E, Example l2, close the spacing between "methyl" and "6- (D-wamino";ii. l 9 Example 2 O, close the spacing between "methyl" and "6-(D-d-amino"; Col 19, line 49, before "dimethyl", insert dry CO1 l lineE3, "57 5%" should be 57 a 7% Tl, 2F, line l6, "wre" should be were o lI, Example 24 close the spacing between "ethyl" and "6- (Db-amino"; ColZl line 23 "Blactum" should be B-lactam t W. K, line F6, "Acetoxyetyl"should be Acetoxyethyl is .71 and 5 Example 26, close the spacingbetween "acetoxyetyl" and "6-(Ddamino";

UNITED STATES PATENT AND TRADEMARK OFFICE CE'HEIGATE @F QORRECTlON 4PATENT NO. 3 873 5Z1 DATED 2 March 25, 1975 lN\/ ENTOR(S) 1 Bertil AkeEkstrom et al It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

Col. Q Example 21, close the spacing between "ethyl" and "6- (Dor-amino"; 2 g Example 28 close the spacing between "ethyl" and "6-(D-oramino"; o l. 2 3, Example 2 9 close the spacing between "ethyl" andC 0l 22, Example so, close the spacing between "ethyl" and "6- ("g andbetween "60" and "amino"; Col. 23, line 18, "chloroformiate" should bechloroformate EFT 3, line E, 'mainteined" should be maintained E 3, line6 O "6- (60" should be 6- (or 9o 24 Example i1 close the spacing between"ethyl" and "6- (or-amino"; gl 2 4, Example 2 close the spacing between"ethyl" and 6-(Dor-amino"; Col Q, line Z, "hydrochloric" should behydrochloride CHI. 24 line 25 "aminophenylacetamino" should beaminophenylacetamido Col 24, line 27, "penicillante" should bepenicillanate E E, 9th item in last column of table, "54 7" should beCol. 27, line 6, 6-(D-oramin0" should be 6-(D-oramino Coll. Z, l i r 1 el 3, "l missing before "Ethoxy etc.";

Col 27, lines 26 and 27 For filling in capsules the followingformulations were made:" should be a separate paragraph in larger type;

Col H, line 5 in the formula, extreme right, "R should be Signed andficalcd this ninth Day 0? December 1975 {STEAM Arrest:

RUHH c. MA$ON C. MARSHALL DANN Arresting Officer Commissioner ufParentsand Trademarks

1. A COMPOUND OF THE STRUCTURAL FORMULA
 2. A compound according to claim1 having the structural formula
 3. A compound according to claim 1having the structural formula
 4. A compound according to claim 1 havingthe structural formula
 5. A compound according to claim 1 having thestructural formula
 6. A compound according to claim 1 having thestructural formula
 7. A compound according to claim 1 having thestructural formula
 8. A compound according to claim 1 having thestructural formula
 9. A compound according to claim 1 having thestructural formula
 10. A compound according to claim 1 having thestructural formula
 11. A compound according to claim 1 having thestructural formula
 12. A compound according to claim 1 having thestructural formula
 13. A compound according to claim 1 having thestructural formula
 14. A compound according to claim 1 wherein R1 is